Liposomal formulations of anthracycline agents and cytidine analogs

ABSTRACT

Compositions which comprise an anthracycline agent, and a cytidine analog are encapsulated in liposomal carriers. The preferred anthracycline agent is selected from the group of daunorubicin, doxorubicin, and idarubicin, while the preferred cytidine analog is selected from the group of cytarabine, gemcitabine, or 5-azacytidine. The combination of the anthracycline agent and cytidine analog encapsulated in said liposomal carriers are useful in achieving a drug retention and a sustained drug release for each therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/587,112, now allowed and having an international filing date of 22Apr. 2005, which is the national phase of PCT applicationPCT/CA2005/000625 having an international filing date of 22 Apr. 2005,which claims benefit under 35 U.S.C. §119(e) to provisional application60/565,210 filed Apr. 22, 2004, The contents of these documents areincorporated herein by reference.

TECHNICAL FIELD

The invention relates to compositions and methods for improved deliveryof combinations of therapeutic agents. More particularly, the inventionconcerns delivery systems which provide combinations of anthracyclineagents and cytidine analogs and derivatives thereof.

BACKGROUND ART

The progression of many life-threatening diseases such as cancer, AE)S,infectious diseases, immune disorders and cardiovascular disorders areinfluenced by multiple molecular mechanisms. Due to this complexity,achieving cures with a single agent has been met with limited success.Thus, combinations of agents have often been used to combat disease,particularly in the treatment of cancers. It appears that there is astrong correlation between the number of agents administered and curerates for cancers such as acute lymphocytic leukemia and metastaticcolorectal cancer (Frei, et al., Clin. Cancer Res. (1998) 4:2027-2037;Fisher, M. D.; Clin Colorectal Cancer (2001) August; 1(2):85-6).

Anthracycline antibiotics such as daunorubicin, doxorubicin, epirubicinand their derivatives comprise known antineoplastic agents.Daunorubicin-based drugs, such as daunorubicin hydrochloride, areprimarily employed because they intercalate with DNA, affecting variousfunctions of the DNA, including DNA and RNA synthesis. They exhibitactivity against acute lymphocytic leukemia, acute granulocyticleukemia, acute myelocytic leukemia, the acute phase of chronicmyelocytic leukemia, and acute nonlymphocytic leukemia. Doxorubicin hasbeen shown effective in the treatment of acute leukemias, malignantlymphomas and selected solid tumors such as breast cancer tumors.Idarubicin exhibits similar activity to these antimetabolites and hasbeen used together with cytarabine for adverse karyotype, acute myeloidleukemia (AML). Giles, F. J., et al., J. Clin. Oncol. (2003)21(9):1722-7.

Cytidine analogs, such as three examples of such analogs includingcytarabine, 5-Azacytidine, and gemcitabine, are known antineoplasticagents. For example, these compounds have demonstrated effectiveness atinhibiting DNA synthesis in leukemia and cancer cells. These propertieshave enabled these compounds to effectively treat acute myelocyticleukemia, acute lymphoblastic leukemia and myelodysplastic syndromes,pancreatic cancer and lung cancer.

US 2004/0052864 discusses the administration of a nonencapsulated DNAmethylation inhibitor and a nonencapsulated anti-neoplastic agent,either singularly or in a free drug cocktail, for the treatment ofdiseases associated with abnormal cell proliferation. However, nopharmaceutical preparations designed to control delivery or half-livesof the drugs were suggested in this publication.

Similarly, U.S. Pat. No. 5,736,155 discusses the preparation of liposomeencapsulated neoplastic agents. Single and multiple antineoplasticagents are contemplated as administered simultaneously or sequentially,however, the combination of an anthracycline antibiotic together with acytidine analog was not suggested.

There are various drawbacks that limit the therapeutic use of drugcocktails. For instance, administration of free drug cocktails oftenresults in rapid clearance of one or all of the drugs before reachingthe tumor site. For this reason, many drugs have been incorporated intodelivery vehicles designed to ‘shield’ them from mechanisms that wouldotherwise result in their clearance from the bloodstream. It is wellknown that liposomes have the ability to provide this ‘shielding’ effectand they are thus able to extend the half-life of therapeutic agents.However, formulation of specific drugs or more than one drug intodelivery vehicles has proven to be difficult because the lipidcomposition of the vehicle often differentially affects thepharmacokinetics of individual drugs. Thus a composition that issuitable for retention and release of one drug may not be suitable forthe retention and release of a second drug.

Investigators of the present invention have identified particulardelivery vehicle formulations required to accommodate a combination ofan anthracycline and a cytidine analog (including daunorubicin andcytarabine-based derivatives), which result in superior drug retentionand sustained drug release of each agent. They have further demonstratedthat synergistic ratios of these drugs, when encapsulated in liposomes,can be successfully maintained in the blood compartment over timeresulting in enhanced efficacy compared to the free drug cocktail andindividual liposomal drugs.

DISCLOSURE OF THE INVENTION

The invention relates to compositions and methods for administeringeffective amounts of anthracycline and cytidine analog (e.g.,daunorubicin, doxorubicin or idarubicin with cytarabine, 5-Azacytidineor gemcitabine) drug combinations using liposomal delivery vehicles thatare stably associated therewith at least one anthracycline agent and onecytidine analog-based drug. These compositions allow the two or moreagents to be delivered to the disease site in a coordinated fashion,thereby assuring that the agents will be present at the disease site ata desired ratio. This result will be achieved whether the agents areco-encapsulated in a lipid-based delivery vehicle, or are encapsulatedin a separate lipid-based delivery vehicles administered such thatdesired ratios are maintained at the disease site. The pharmacokinetics(PK) of the composition are controlled by the lipid-based deliveryvehicles themselves such that coordinated delivery is achieved (providedthat the PK of the delivery systems are comparable).

Thus, in one aspect, the invention provides a liposome composition forparenteral administration comprising at least one anthracycline and onecytidine analog associated with the liposomes at therapeuticallyeffective ratios especially those that are non-antagonistic. Thetherapeutically effective non-antagonistic ratio of the agents isdetermined by assessing the biological activity or effects of the agentson relevant cell culture or cell-free systems, as well as tumorhomogenates from individual patient biopsies, over a range ofconcentrations. Frequent combinations are daunorubicin with cytarabine,among other cytidine analogs together with daunorubicin, doxorubicin ortheir derivatives. Also frequently, a combination is provided comprisingcytarabine (or another cytidine analog) and an anthracycline comprisingdaunorubicin, doxorubicin or idarubicin, among other knownanthracyclines. Any method which results in determination of a ratio ofagents which maintains a desired therapeutic effect may be used.

The composition comprises at least one anthracycline and one cytidineagent in a mole ratio of the anthracycline to the cytidine agent whichexhibits a desired biologic effect to relevant cells in culture,cell-free systems or tumor homogenates. Preferably, the ratio is that atwhich the agents are non-antagonistic. By “relevant” cells, applicantsrefer to at least one cell culture or cell line which is appropriate fortesting the desired biological effect. As these agents are used asantineoplastic agents, “relevant” cells are those of cell linesidentified by the Developmental Therapeutics Program (DTP) of theNational Cancer Institute (NCI)/National Institutes of Health (NIH) asuseful in their anticancer drug discovery program. Currently the DTPscreen utilizes 60 different human tumor cell lines. The desiredactivity on at least one of such cell lines would need to bedemonstrated. By “tumor homogenate,” the applicant refers to cellsgenerated from the homogenization of patient biopsies or tumors.Extraction of whole tumors or tumor biopsies can be achieved throughstandard medical techniques by a qualified physician and homogenizationof the tissue into single cells can be carried out in the laboratoryusing a number of methods well-known in the art.

In another aspect, the invention is directed to a method to deliver atherapeutically effective amount of an anthracycline:cytidine analogcombination (e.g., daunorubicin:cytarabine) to a desired target byadministering the compositions of the invention.

The invention is also directed to a method to deliver a therapeuticallyeffective amount of an anthracycline:cytidine analog combination byadministering an anthracycline stably associated with a first deliveryvehicle and a cytidine analog stably associated with a second deliveryvehicle. The first and second delivery vehicles may be contained inseparate vials, the contents of the vials being administered to apatient simultaneously or sequentially. In one embodiment, the ratio ofthe anthracycline and the cytidine analog is non-antagonistic.

In another aspect, the invention is directed to a method to prepare atherapeutic composition comprising liposomes containing a ratio of atleast one anthracycline and one cytidine analog which provides a desiredtherapeutic effect which method comprises providing a panel of at leastone anthracycline and one cytidine analog wherein the panel comprises atleast one, but preferably a multiplicity of ratios of said drugs,testing the ability of the members of the panel to exert a biologicaleffect on a relevant cell culture, cell-free system or tumor homogenateover a range of concentrations, selecting a member of the panel whereinthe ratio provides a desired therapeutic effect on said cell culture,cell-free system or tumor homogenate over a suitable range ofconcentrations; and stably associating the ratio of drugs represented bythe successful member of the panel into lipid-based drug deliveryvehicles. In preferred embodiments, the abovementioned desiredtherapeutic effect is non-antagonistic.

As further described below, in a preferred embodiment, in designing anappropriate combination in accordance with the method described above,the non-antagonistic ratios are selected as those that have acombination index (CI) of ≦1.1. In further embodiments, suitableliposomal formulations are designed such that they stably incorporate aneffective amount of an anthracycline:cytidine analog combination (e.g.,daunorubicin:cytarabine) and allow for the sustained release of bothdrugs in vivo. Preferred formulations contain at least one negativelycharged lipid, such as phosphatidylglycerol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the combination index (CI) plotted as afunction of the fraction of P388 murine lymphocytic leukemia cellsaffected (f_(a)) by combinations of daunorubicin:cytarabine (or Ara-C)at various mole ratios: 10:1 (squares), 5:1 (triangles), 1:1 (hollowcircles), 1:5 (inverted triangles) and 1:10 (filled-in circles).

FIG. 1B is a graph showing the combination (CI) plotted as a function ofthe fraction of L1210 murine lymphocytic leukemia cells affected (f_(a))by combinations of daunorubicin:cytarabine (or Ara-C) at various moleratios: 10:1 (squares), 5:1 (triangles), 1:1 (diamonds), 1:5 (invertedtriangles) and 1:10 (circles).

FIG. 1C is a graph showing the CI versus various mole ratios ofdaunorubicin:cytarabine (10:1, 5:1, 1:1, 1:5, 1:10) in P388 murinelymphocytic leukemia cells. CI values at drug concentrations sufficientto cause 75% (ED75) and 90% (ED90) tumor cell growth inhibition arecompared at the different daunorubicin:cytarabine molar ratios.

FIG. 1D is a graph showing the CI versus various mole ratios ofdaunorubicin:cytarabine (10:1, 5:1, 1:1, 1:5, 1:10) in L1210 murinelymphocytic leukemia cells. CI values at drug concentrations sufficientto cause 75% (ED75) and 90% (ED90) tumor cell growth inhibition arecompared at the different daunorubicin:cytarabine molar ratios.

FIG. 2A is a graph showing the plasma elimination curves fordaunorubicin and cytarabine at various time points after intravenousadministration to BDF-1 mice in DSPC:DSPG:CHOL (7:2:1 mol ratio)liposomes.

FIG. 2B is a graph of the daunorubicin:cytarabine ratio (mol:mol) in theplasma as a function of time after intravenous administration ofdaunorubicin:cytarabine (about 1:5 molar ratio) dual-loadedDSPC:DSPG:CHOL (7:2:1 mol ratio) liposomes. Data points represent themolar ratios of daunorubicin:cytarabine determined in plasma(+/−standard deviation) at the specified time points.

FIG. 3A is a graph showing the efficacy of co-loaded liposome entrappeddaunorubicin:cytarabine (about 1:5 mol ratio) compared to individualliposomal encapsulated drugs and free drug cocktail administered viai.v. (Q3DX3) against the P388 lymphocytic leukemia model in mice. Micewere organized into appropriate treatment groups consisting of controland treatment groups including saline (circles), liposomal daunorubicin(triangles), liposomal cytarabine (squares), free cocktail ofdaunorubicin:cytarabine (9:600 mg/kg) (inverted triangles) anddaunorubicin:cytarabine co-loaded in DSPC:DSPG:Chol (7:2:1, mol:mol)liposomes resulting in a final daunorubicin:cytarabine molar ratio ofabout 1:5 (diamonds).

FIG. 3B is a graph showing the efficacy of co-loaded liposome entrappeddaunorubicin:cytarabine (about 1:5 mol ratio) compared to individualliposomal encapsulated drugs and free cocktail administered via i.v.(Q3DX3) against the L1210 lymphocytic leukemia model in mice. Mice wereorganized into appropriate treatment groups consisting of control andtreatment groups including saline (circles), liposomal daunorubicin(triangles), liposomal cytarabine (squares), free cocktail ofdaunorubicin:cytarabine (12:30 mg/kg) (open diamonds) anddaunorubicin:cytarabine co-loaded in DSPC:DSPG:Chol (7:2:1, mol:mol)liposomes resulting in a final daunorubicin:cytarabine molar ratio ofabout 1:5 (filled diamonds).

MODES OF CARRYING OUT THE INVENTION

Unless defined otherwise, all terms of art, notations and otherscientific terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisinvention belongs. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. All patents,applications, published applications and other publications referred toherein are incorporated by reference in their entirety. If a definitionset forth in this section is contrary to or otherwise inconsistent witha definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.¹ ¹AbbreviationsDSPC: distearoylphosphatidylcholine; PG: phosphatidylglycerol; DSPG:distearoylphosphatidylglycerol; PI: phosphatidylinositol; SM:sphingomyelin; Chol or CH: cholesterol; CHE: cholesteryl hexadecylether; SUV: small unilamellar vesicle; LUV: large unilamellar vesicle;MLV: multilamellar vesicle; MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide; EDTA:ethylenediaminetetraacetic acid; HEPES:N[2-hydroxylethyl]-piperazine-N-[2-ethanesulfonic acid]; MS: HEPESbuffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4); SHE: 300 mM sucrose,20 mM HEPES, 30 mM EDTA; TEA: triethanolamine; CI: combination index;f_(a): fraction affected.

As used herein, “a” or “an” means “at least one” or “one or more.”

The invention provides compositions comprising liposomes stablyassociated therewith at least one anthracycline antibiotic (e.g.,daunorubicin) and one cytosine analog agent (e.g., cytarabine), whereinthe anthracycline antibiotic and cytosine analog agent are present atanthracycline antibiotic:cytosine analog agent (e.g.,daunorubicin:cytarabine) mole ratios that exhibit a desired cytotoxic,cytostatic or biologic effect to relevant cells or tumor homogenates.

Preferably, liposomal compositions provided herein will includeliposomes stably associated therewith at least one anthracycline and onecytidine agent in a mole ratio of the anthracycline:cytidine analogwhich exhibits a non-antagonistic effect to relevant cells or tumorhomogenates.

In further embodiments of the invention, the above described lipid-baseddelivery vehicles comprise a third or fourth agent. Any therapeutic,diagnostic or cosmetic agent may be included.

In one aspect of the invention, liposomes which comprisephosphatidylcholine are provided, preferablydistearoylphosphatidylcholine. In another aspect of the invention,liposomes which comprise a sterol are provided. Preferably the sterol ischolesterol.

The lipid-based delivery vehicles of the present invention may be usednot only in parenteral administration but also in topical, nasal,subcutaneous, intraperitoneal, intramuscular, aerosol or oral deliveryor by the application of the delivery vehicle onto or into a natural orsynthetic implantable device at or near the target site for therapeuticpurposes or medical imaging and the like. Preferably, the lipid-baseddelivery vehicles of the invention are used in parenteraladministration, most preferably, intravenous administration.

The preferred embodiments herein described are not intended to beexhaustive or to limit the scope of the invention to the precise formsdisclosed. They are chosen and described to best explain the principlesof the invention and its application and practical use to allow othersskilled in the art to comprehend its teachings.

Cytidine Analogs

Antimetabolites or, more particularly, cytidine analogs such ascytarabine, 5-Azacytidine, and gemcitabine (2′,2′-Difluorodeoxycytidine)are known antineoplastic agents. Cytidine analogs may also be referredto in the art as cytosine nucleoside analogs. Antimetabolites arecompounds that are similar enough to a natural chemical to participatein a normal biochemical reaction in cells but different enough tointerfere with the normal division and functions of cells. Thesecompounds generally inhibit a normal metabolic process.

Cytarabine is a pyrimidine nucleoside antimetabolite. This compound isan analog of 2′-deoxycytidine with the 2′-hydroxyl in a position transto the 3′-hydroxyl of the sugar. Cytarabine is considered equivalentwith 4-Amino-1-β-d-arabinofuranosyl-2(1H)-pyrimidinone,1-β-d-arabinofuranosylcytosine, Ara-C, β-cytosine arabinoside,aracytidine, CHX-3311, U-19920, Alexan, Arabitin, Aracytine, Cytarbel,Cytosar, Erpalfa, Iretan and Udicil. In cytidine analogs such ascytarabine, the sugar moiety comprises an arabinose rather than ribose.Cytarabine is recognized as useful in the therapy of acute myelocyticleukemia (AML) and has proven effectiveness in the remission of thisdisorder. However, the mechanism of action of cytarabine is uncertain,nevertheless incorporation of this nucleotidase into DNA leads to aninhibition of polymerization by termination of strand synthesis.

Cytarabine must be “activated” via conversion of the 5-monophosphatenucleotide (AraCMP) to terminate strand synthesis. AraCMP is then ableto react with selected nucleotide kinases to form diphosphate andtriphosphate nucleotides (AraCDP and AraCTP). Cytarabine incorporationinto DNA is S-phase specific, thus dosing has been advocated over atleast one full cell cycle to obtain inhibition of DNA synthesis.Inhibition of DNA synthesis occurs at low AraCTP concentrations andinhibits DNA chain elongation by incorporation of AraC into the terminalportion of a growing DNA chain. Moreover, there appears to be acorrelation between the amount of AraC incorporated into the chain andthe inhibition of DNA synthesis.

Subjects can develop resistance to cytarabine. Such resistance isgenerally due to a deficiency of deoxycytidine kinase, which producesAraCMP. In addition, degrative enzymes such as cytidine deaminase (whichdeaminates AraC to nontoxic arauridine) and dCMP (which converts AraCMPto inactive AraUMP) also affect efficacy.

A drawback of cytarabine is its toxicity. This compound is a potentmyleosuppresive agent capable of producing severe leucopenia,thrombocytopenia and anemia with notable megaloblastic changes.Gastrointestinal disturbances, fever, conjunctivitis, pneumonitis,hepatic dysfunction, dermatitis, and neurotoxic side effects have alsobeen noted generally when higher doses are administered.

5-Azacytidine (Azacytidine; 5-AzaC) is a compound that exhibitsantineoplastic activity. This compound is known as useful for thetreatment of AML, acute lymphoblastic leukemia and myelodysplasticsyndromes. Current studies are evaluating the effects of this compoundin beta thalassemia, acute myeloid leukemia, myelodysplastic syndrome,advanced or metastatic solid tumors, non-Hodgkin's lymphoma, multiplemyeloma, non-small cell lung cancer and prostate cancer. 5-AzaC has beenshown to inhibit DNA methylation, which in turn affects gene expression.Side effects include decreased white and red blood cell and plateletcount, nausea, vomiting, fatigue, diarrhea, among other effects.

Gemcitabine is a nucleoside analog that exhibits antitumor activity.Gemcitabine HCl consists of a 2′-deoxy-2′,2′-difluorocytidinemonohydrochloride (0-isomer) and is known as effective in treatingpancreatic cancer and lung cancer. In general, gemcitabine preventscells from making DNA and RNA by interfering with the synthesis ofnucleic acids. This action stops the growth of cancer cells, causing thecells to die. Side effects include decreased white blood cell andplatelet count, nausea, vomiting, fatigue, diarrhea, flu-like symptoms,rashes, among other effects.

Anthracycline Antibiotics

Anthracycline antibiotics such as daunorubicin and doxorubicin and theirderivatives comprise known antineoplastic agents produced by the fungusStreptomyces peucetius. Idarubicin (4-demethoxydaunorubicin) comprises asynthetic derivative of daunorubicin lacking the methoxy group on C4 ofthe aglycone ring. These compounds intercalate with DNA, affectingvarious functions of the DNA, including DNA and RNA synthesis. Theinteraction with DNA generally causes single and/or double strand breaksand sister chromatid exchange. One particular pharmaceutical form ofdaunorubicin (daunorubicin hydrochloride) has been demonstrated toprevent cell division in doses that do not interfere with nucleic acidsynthesis. The mechanism by which the scission of the DNA isaccomplished is not fully understood, but it is believed to be mediatedby the action of topoisomerase II or by the generation of free radicals.Moreover, anthracyclines are also known to interact with cell membranesand alter their functions, which may play a role in their antitumoractions cardiotoxicity.

Daunorubicin (daunomycin, rubidomycin, leukaemomycin C, RP-13057,CERUBIDINE®) is known as useful in the treatment of acute lymphocyticleukemia, acute granulocytic leukemia, acute myelocytic leukemia, theacute phase of chronic myelocytic leukemia, and acute nonlymphocyticleukemia. In addition, this compound has been demonstrated to have someactivity in solid tumors and against lymphomas. Daunorubicin is aglycoside formed by a tatrasysclic agycone-daunomycinone, and an aminosugar-daunosamine. Oral absorption of daunorubicin is low, and it ismost frequently administered intravenously. The half-life ofdistribution is 45 minutes and 19 hours for elimination. Daunorubicin iseliminated via conversion to a less active form, daunorubicinol.Daunorubicin and its derivatives have certain toxicities such as bonemarrow depression, stomatitis, alopecia, gastrointestinal disturbances,cardiac dysrhythmias, pulmonary edema. The most widely recognizeddrawback of these compositions is the potential for cardiomyopathy inacute or chronic forms which can quickly become a life-threateningsituation. Cardiotoxicity, in particular, manifests itself as congestiveheart failure in 15-40% of patients undergoing therapy. Generally, suchside effects are due to the dosage utilized, with the occurrenceincreasing at higher does. It has been recognized, however, thatconcomitant administration of dexrazoxane (ADR-529) or amifostine(WR-2721 or WP-1065) will reduce cardiac damage caused by thesecompositions. Moreover, there is evidence to suggest that cardiac damageduring anthracycline therapy can be reduced by simultaneousadministration of the iron chelators such as dipyridoxyl andaminopolycarboxylic acid based chelating agents, and their metalchelates. See U.S. Pat. No. 6,147,094.

Doxorubicin (adriamycin, rubrex, 12-naphthacenedione,14-hydroxydaunomycin, NSC-123127) differs from daunorubicin only inhaving a hydroxyacetyl group in place of the acetyl group indaunorubicin, in position 8. Doxorubicin has been shown effective in thetreatment of acute leukemias, malignant lymphomas and selected solidtumors such as breast cancer tumors. This composition has been utilizedtogether with cyclophosphamide, vincristine and procarbazine in thetreatment of Hodgkin's disease and non-Hodgkin's lymphoma. Additionaltherapeutic utilities have been demonstrated for sarcomas, plasma cellmyeloma, metastatic thyroid carcinoma, gastric carcinoma, broncheogeniccarcinoma, transitional cell carcinoma, and carcinomas of the ovary,endometrium, thyroid, testes and cervix. The toxicities of doxorubicinare similar to daunorubicin set out above. Analogs of doxorubicin, suchas epirubicin (4′-epidoxorubicin), morpholino derivatives and relatedanthracenedione mitoxantrone, have been shown to have less cardiactoxicity with high clinical activity.

Determining Non-Antagonistic Daunorubicin:Cytarabine Ratios In Vitro

In a further aspect of the invention anthracycline agents and cytidineanalogs will be encapsulated into liposomes at synergistic or additive(i.e. non-antagonistic) ratios. Determination of ratios of agents thatdisplay synergistic or additive combination effects may be carried outusing various algorithms, based on the types of experimental datadescribed below. These methods include isobologram methods (Loewe, etal., Arzneim-Forsch (1953) 3:285-290; Steel, et al., Int. J. Radiol.Oncol. Biol. Phys. (1979) 5:27-55), the fractional product method (Webb,Enzyme and Metabolic Inhibitors (1963) Vol. 1, pp. 1-5. New York:Academic Press), the Monte Carlo simulation method, CombiTool, ComboStatand the Chou-Talalay median-effect method based on an equation describedin Chou, J. Theor. Biol. (1976) 39:253-276; and Chou, Mol. Pharmacol.(1974) 10:235-247). Alternatives include surviving fraction (Zoli, etal., Int. J. Cancer (1999) 80:413-416), percentage response togranulocyte/macrophage-colony forming unit compared with controls(Pannacciulli, et al., Anticancer Res. (1999) 19:409-412) and others(Berenbaum, Pharmacol. Rev. (1989) 41:93-141; Greco, et al., PharmacolRev. (1995) 47:331-385).

The Chou-Talalay median-effect method is preferred. The analysisutilizes an equation wherein the dose that causes a particular effect,f_(a), is given by:D=D _(m) [f _(a)/(1−f _(a))]^(1/m)in which D is the dose of the drug used, f_(a) is the fraction of cellsaffected by that dose, D_(m) is the dose for median effect signifyingthe potency and m is a coefficient representing the shape of thedose-effect curve (m is 1 for first order reactions).

This equation can be further manipulated to calculate a combinationindex (CI) on the basis of the multiple drug effect equation asdescribed by Chou and Talalay, Adv. Enzyme Reg. (1984) 22:27-55; and byChou, et al., in: Synergism and Antagonism in Chemotherapy, Chou andRideout, eds., Academic Press: New York 1991:223-244. A computer program(CalcuSyn) for this calculation is found in Chou and Chou (“Dose-effectanalysis with microcomputers: quantitation of ED50, LD50, synergism,antagonism, low-dose risk, receptor ligand binding and enzyme kinetics”:CalcuSyn Manual and Software; Cambridge: Biosoft 1987).

The combination index equation is based on the multiple drug-effectequation of Chou-Talalay derived from enzyme kinetic models. An equationdetermines only the additive effect rather than synergism andantagonism. However, according to the CalcuSyn program, synergism isdefined as a more than expected additive effect, and antagonism as aless than expected additive effect. Chou and Talalay in 1983 proposedthe designation of CI=1 as the additive effect, thus from the multipledrug effect equation of two drugs, we obtain:CI=(D)₁/(D _(x))₁+(D)₂/(D _(x))₂  [Eq. 1]for mutually exclusive drugs that have the same or similar modes ofaction, and it is further proposed thatCI=(D)₁/(D _(x))+(D)₂/(D _(x))₂+((D)₁(D)₂)/(D _(x))₁(D _(x))₂  [Eq. 2]for mutually non-exclusive drugs that have totally independent modes ofaction. CI<1, =1, and >1 indicates synergism, additive effect, andantagonism, respectively. Equation 1 or equation 2 dictates that drug 1,(D)₁, and drug 2, (D)₂, (in the numerators) in combination inhibit x %in the actual experiment. Thus, the experimentally observed x %inhibition may not be a round number but most frequently has a decimalfraction. (D_(x))₁ and (D_(x))₂ (in the denominators) of equations 1 and2 are the doses of drug 1 and drug 2 alone, respectively, inhibiting x%.

For simplicity, mutual exclusivity is usually assumed when more than twodrugs are involved in combinations (CalcuSyn Manual and Software;Cambridge: Biosoft 1987).

A two-drug combination may be further used as a single pharmaceuticalunit to determine synergistic or additive interactions with a thirdagent. In addition, a three-agent combination may be used as a unit todetermine non-antagonistic interactions with a fourth agent, and so on.

The underlying experimental data are generally determined in vitro usingcells in culture or cell-free systems. Preferably, the combination index(CI) is plotted as a function of the fraction of cells affected (f_(a))as shown in FIGS. 1A and 1B which, as explained above, is a surrogateparameter for concentration range. Preferred combinations of agents arethose that display synergy or additivity over a substantial range off_(a) values. Combinations of agents are selected if non-antagonisticover at least 5% of the concentration range wherein greater than 1% ofthe cells are affected, i.e., an f_(a) range greater than 0.01.Preferably, a larger portion of overall concentration exhibits afavorable CI; for example, 5% of an f_(a) range of 0.2-1.0. Morepreferably 10% of this range exhibits a favorable CI. Even morepreferably, 20% of the f_(a) range, preferably over 50% and mostpreferably over at least 70% of the f_(a) range of 0.2 to 1.0 areutilized in the compositions. Combinations that display synergy over asubstantial range of f_(a) values may be re-evaluated at a variety ofagent ratios to define the optimal ratio to enhance the strength of thenon-antagonistic interaction and increase the f_(a) range over whichsynergy is observed.

While it would be desirable to have synergy over the entire range ofconcentrations over which cells are affected, it has been observed thatin many instances, the results are considerably more reliable in anf_(a) range of 0.2-0.8 when using a spectrophotometric method such asthe MTT assay detailed in Example 1. Thus, although the synergyexhibited by combinations of the invention is set forth to exist withinthe broad range of 0.01 or greater, it is preferable that the synergy beestablished in the f_(a) range of 0.2-0.8. Other more sensitive assays,however, can be used to evaluate synergy at f_(a) values greater than0.8, for example, bioluminescence or clonogenecity assays.

The optimal combination ratio may be further used as a singlepharmaceutical unit to determine synergistic or additive interactionswith a third agent. In addition, a three-agent combination may be usedas a unit to determine non-antagonistic interactions with a fourthagent, and so on.

As set forth above, the in vitro studies on cell cultures will beconducted with “relevant” cells. The choice of cells will depend on theintended therapeutic use of the agent. Only one relevant cell line orcell culture type need exhibit the required non-antagonistic effect inorder to provide a basis for the compositions to come within the scopeof the invention.

For example, in one preferred embodiment of the invention, thecombination of agents is intended for anticancer therapy. In a frequentembodiment, the combination of agents is intended for leukemia orlymphoma therapy. Appropriate choices will then be made of the cells tobe tested and the nature of the test. In particular, tumor cell linesare suitable subjects and measurement of cell death or cell stasis is anappropriate end point. As will further be discussed below, in thecontext of attempting to find suitable non-antagonistic combinations forother indications, other target cells and criteria other thancytotoxicity or cell stasis could be employed.

For determinations involving antitumor agents, cell lines may beobtained from standard cell line repositories (NCI or ATCC for example),from academic institutions or other organizations including commercialsources. Preferred cell lines would include one or more selected fromcell lines identified by the Developmental Therapeutics Program of theNCI/NIH. The tumor cell line screen used by this program currentlyidentifies 60 different tumor cell lines representing leukemia,melanoma, and cancers of the lung, colon, brain, ovary, breast, prostateand kidney. The required non-antagonistic effect over a desiredconcentration range need be shown only on a single cell type; however,it is preferred that at least two cell lines exhibit this effect, morepreferably three cell lines, more preferably five cell lines, and morepreferably 10 cell lines. The cell lines may be established tumor celllines or primary cultures obtained from patient samples. The cell linesmay be from any species but the preferred source will be mammalian andin particular human. The cell lines may be genetically altered byselection under various laboratory conditions, and/or by the addition ordeletion of exogenous genetic material. Cell lines may be transfected byany gene-transfer technique, including but not limited to, viral orplasmid-based transfection methods. The modifications may include thetransfer of cDNA encoding the expression of a specific protein orpeptide, a regulatory element such as a promoter or enhancer sequence orantisense DNA or RNA. Genetically engineered tissue culture cell linesmay include lines with and without tumor suppressor genes, that is,genes such as p53, pTEN and p16; and lines created through the use ofdominant negative methods, gene insertion methods and other selectionmethods. Preferred tissue culture cell lines that may be used toquantify cell viability, e.g., to test antitumor agents, include, butare not limited to, P388, L1210, HL-60, MOLT-4, KBM-3, WeHi-3, H460,MCF-7, SF-268, HT29, HCT-116, LS180, B16-F10, A549, Capan-1, CAOV-3,IGROV1, PC-3, MX-1 and MDA-MB-231.

In one preferred embodiment, the given effect (f_(a)) refers to celldeath or cell stasis after application of a cytotoxic agent to a cellculture. Cell death or viability may be measured, for example, using thefollowing methods:

CYTOTOXICITY ASSAY REFERENCE MTT assay Mosmann, J. Immunol. Methods(1983) 65(1-2): 55-63. Trypan blue dye exclusion Bhuyan, et al.,Experimental Cell Research (1976) 97: 275-280. Radioactive tritium (³H)-Senik, et al., Int. J. Cancer thymidine incorporation or (1975) 16(6):946-959. DNA intercalating assay Radioactive chromium-51 Brunner, etal., Immunology release assay (1968) 14: 181-196. Glutamate pyruvateMitchell, et al., J. of Tissue Culture Methods transaminase, creatine(1980) 6(3&4): 113-116. phosphokinase and lactate dehydrogenase enzymeleakage Neutral red uptake Borenfreund and Puerner, Toxicol. Lett.(1985) 39: 119-124. Alkaline phosphatase Kyle, et al., J. Toxicol.Environ. Health activity (1983) 12: 99-117. Propidium iodide stainingNieminen, et al., Toxicol. Appl. Pharmacol. (1992) 115: 147-155.Bis-carboxyethyl- Kolber, et al., J. Immunol. Methods carboxyfluorescein(1988) 108: 255-264. (BCECF) retention Mitochondrial Johnson, et al.,Proc. Natl. Acad. Sci. USA membrane potential (1980) 77: 990-994.Clonogenic Assays Puck, et al., J. of Experimental Medicine (1956) 103:273-283. LIVE/DEAD Viability/ Morris, Biotechniques (1990) 8: 296-308.Cytotoxicity assay Sulforhodamine B Rubinstein, et al., J. Natl. CancerInstit. (SRB) assays (1990) 82: 1113-1118.

Non-antagonistic ratios of two or more agents can be determined fordisease indications other than cancer and this information can be usedto prepare therapeutic formulations of two or more drugs for thetreatment of these diseases. With respect to in vitro assays, manymeasurable endpoints can be selected from which to define drug synergy,provided those endpoints are therapeutically relevant for the specificdisease.

As set forth above, the in vitro studies on cell cultures will beconducted with “relevant” cells. The choice of cells will depend on theintended therapeutic use of the agent. In vitro studies on individualpatient biopsies or whole tumors can be conducted with “tumorhomogenate,” generated from homogenization of the tumor sample(s) intosingle cells.

In one preferred embodiment, the given effect (f_(a)) refers to celldeath or cell stasis after application of a cytotoxic agent to a“relevant” cell culture or “tumor homogenate” (see Example 1). Celldeath or viability may be measured using a number of methods known inthe art.

Preparation of Lipid-Based Delivery Vehicles

Preferred lipid carriers for use in this invention are liposomes.Liposomes can be prepared as described in Liposomes: Rational Design (A.S. Janoff, ed., Marcel Dekker, Inc., New York, N.Y.), or by additionaltechniques known to those knowledgeable in the art. Suitable liposomesfor use in this invention include large unilamellar vesicles (LUVs),multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) andinterdigitating fusion liposomes.

Liposomes for use in this invention may be prepared to contain aphosphatidylcholine lipid, such as distearylphosphatidylcholine.Liposomes of the invention may also contain a sterol, such ascholesterol. Liposomes may also contain therapeutic lipids, whichexamples include ether lipids, phosphatidic acid, phosphonates, ceramideand ceramide analogs, sphingosine and sphingosine analogs andserine-containing lipids.

Liposomes may also be prepared with surface stabilizing hydrophilicpolymer-lipid conjugates such as polyethylene glycol-DSPE, to enhancecirculation longevity. The incorporation of negatively charged lipidssuch as phosphatidylglycerol (PG) and phosphatidylinositol (PI) may alsobe added to liposome formulations to increase the circulation longevityof the carrier. These lipids may be employed to replace hydrophilicpolymer-lipid conjugates as surface stabilizing agents. Preferredembodiments of this invention may make use of liposomes containingphosphatidylglycerol (PG) or phosphatidylinositol (P1) to preventaggregation thereby increasing the blood residence time of the carrier.

In one embodiment, liposome compositions in accordance with thisinvention are preferably used to treat cancer. Delivery of encapsulateddrugs to a tumor site is achieved by administration of liposomes of theinvention. Preferably liposomes have a diameter of less than 300 nm.Most preferably liposomes have a diameter of less than 200 nm. Tumorvasculature is generally leakier than normal vasculature due tofenestrations or gaps in the endothelia. This allows delivery vehiclesof 200 nm or less in diameter to penetrate the discontinuous endothelialcell layer and underlying basement membrane surrounding the vesselssupplying blood to a tumor. Selective accumulation of the deliveryvehicles into tumor sites following extravasation leads to enhancedanticancer drug delivery and therapeutic effectiveness.

Various methods may be utilized to encapsulate active agents inliposomes. “Encapsulation,” includes covalent or non-covalentassociation of an agent with the lipid-based delivery vehicle. Forexample, this can be by interaction of the agent with the outer layer orlayers of the liposome or entrapment of an agent within the liposome,equilibrium being achieved between different portions of the liposome.Thus encapsulation of an agent can be by association of the agent byinteraction with the bilayer of the liposomes through covalent ornon-covalent interaction with the lipid components or entrapment in theaqueous interior of the liposome, or in equilibrium between the internalaqueous phase and the bilayer. “Loading” refers to the act ofencapsulating one or more agents into a delivery vehicle.

Encapsulation of the desired combination can be achieved either throughencapsulation in separate delivery vehicles or within the same deliveryvehicle. Where encapsulation into separate liposomes is desired, thelipid composition of each liposome may be quite different to allow forcoordinated pharmacokinetics. By altering the vehicle composition,release rates of encapsulated drugs can be matched to allow desiredratios of the drugs to be delivered to the tumor site. Means of alteringrelease rates include increasing the acyl-chain length of vesicleforming lipids to improve drug retention, controlling the exchange ofsurface grafted hydrophilic polymers such as PEG out of the liposomemembrane and incorporating membrane-rigidifying agents such as sterolsor sphingomyelin into the membrane. It should be apparent to thoseskilled in the art that if a first and second drug are desired to beadministered at a specific drug ratio and if the second drug is retainedpoorly within the liposome composition of the first drug (e.g.,DMPC/Chol), that improved pharmacokinetics may be achieved byencapsulating the second drug in a liposome composition with lipids ofincreased acyl chain length (e.g., DSPC/Chol). When encapsulated inseparate liposomes, it should be readily accepted that ratios ofanthracycline agents-to-cytidine analogs that have been determined on apatient-specific basis to provide optimal therapeutic activity can begenerated for individual patients by combining the appropriate amountsof each liposome-encapsulated drug prior to administration.Alternatively, two or more agents may be encapsulated within the sameliposome.

Techniques for encapsulation are dependent on the nature of the deliveryvehicles. For example, therapeutic agents may be loaded into liposomesusing both passive and active loading methods. Passive methods ofencapsulating active agents in liposomes involve encapsulating the agentduring the preparation of the liposomes. This includes a passiveentrapment method described by Bangham, et al. (J. Mol. Biol. (1965)12:238). This technique results in the formation of multilamellarvesicles (MLVs) that can be converted to large unilamellar vesicles(LUVs) or small unilamellar vesicles (SUVs) upon extrusion. Anothersuitable method of passive encapsulation includes an ether injectiontechnique described by Deamer and Bangham (Biochim. Biophys. Acta (1976)443:629) and the Reverse Phase Evaporation technique as described bySzoka and Paphadjopoulos (P.N.A.S. (1978) 75:4194). In addition, anothersuitable method of passive encapsulation involves passive equilibrationafter the formation of liposomes. This process involves incubatingpre-formed liposomes under altered or non-ambient (based on temperature,pressure, etc.) conditions and adding a therapeutic agent (e.g.,cytidine analog or anthracycline agent) to the exterior of theliposomes. The therapeutic agent then equilibrates into the interior ofthe liposomes, across the liposomal membrane. The liposomes are thenreturned to ambient conditions and unencapsulated therapeutic agent, ifpresent, is removed via dialysis or another suitable method.

Active methods of encapsulation include the pH gradient loadingtechnique described in U.S. Pat. Nos. 5,616,341, 5,736,155 and 5,785,987and active metal-loading. One method of pH gradient loading is thecitrate-base loading method utilizing citrate as the internal buffer ata pH of 4.0 and a neutral exterior buffer. Other methods employed toestablish and maintain a pH gradient across a liposome involve the useof an ionophore that can insert into the liposome membrane andtransportions across membranes in exchange for protons (see U.S. Pat.No. 5,837,282). A recent and preferred technique utilizing transitionmetals to drive the uptake of drugs into liposomes via complexation inthe absence of an ionophore may also be used. This technique relies onthe formation of a drug-metal complex rather than the establishment of apH gradient to drive uptake of drug.

Passive and active methods of entrapment may also be coupled in order toprepare a liposome formulation containing more than one encapsulatedagent.

Administering Compositions of the Invention In Vivo

As mentioned above, the delivery vehicle compositions of the presentinvention may be administered to warm-blooded animals, including humansas well as to domestic avian species. For treatment of human ailments, aqualified physician will determine how the compositions of the presentinvention should be utilized with respect to dose, schedule and route ofadministration using established protocols. Such applications may alsoutilize dose escalation should agents encapsulated in delivery vehiclecompositions of the present invention exhibit reduced toxicity tohealthy tissues of the subject.

Preferably, the pharmaceutical compositions of the present invention areadministered parenterally, i.e., intraarterially, intravenously,intraperitoneally, subcutaneously, or intramuscularly. More preferably,the pharmaceutical compositions are administered intravenously orintraperitoneally by a bolus or infusional injection. For example, seeRahman, et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410;Papahadjopoulos, et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat.No. 4,224,179; Lenk, et al., U.S. Pat. No. 4,522,803; and Fountain, etal., U.S. Pat. No. 4,588,578, incorporated by reference.

In other methods, the pharmaceutical or cosmetic preparations of thepresent invention can be contacted with the target tissue by directapplication of the preparation to the tissue. The application may bemade by topical, “open” or “closed” procedures. By “topical”, it ismeant the direct application of the multi-drug preparation to a tissueexposed to the environment, such as the skin, oropharynx, externalauditory canal, and the like. “Open” procedures are those proceduresthat include incising the skin of a patient and directly visualizing theunderlying tissue to which the pharmaceutical preparations are applied.This is generally accomplished by a surgical procedure, such as athoracotomy to access the lungs, abdominal laparotomy to accessabdominal viscera, or other direct surgical approach to the targettissue. “Closed” procedures are invasive procedures in which theinternal target tissues are not directly visualized, but accessed viainserting instruments through small wounds in the skin. For example, thepreparations may be administered to the peritoneum by needle lavage.Alternatively, the preparations may be administered through endoscopicdevices.

Pharmaceutical compositions comprising delivery vehicles of theinvention are prepared according to standard techniques and may comprisewater, buffered water, 0.9% saline, 0.3% glycine, 5% dextrose,iso-osmotic sucrose solutions and the like, including glycoproteins forenhanced stability, such as albumin, lipoprotein, globulin, and thelike. These compositions may be sterilized by conventional, well-knownsterilization techniques. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, and the like. Additionally, the delivery vehicle suspensionmay include lipid-protective agents which protect lipids againstfree-radical and lipid-peroxidative damages on storage. Lipophilicfree-radical quenchers, such as alpha-tocopherol and water-solubleiron-specific chelators, such as ferrioxamine, are suitable.

The concentration of delivery vehicles in the pharmaceuticalformulations can vary widely, such as from less than about 0.05%,usually at or at least about 2-5% to as much as 10 to 30% by weight andwill be selected primarily by fluid volumes, viscosities, and the like,in accordance with the particular mode of administration selected. Forexample, the concentration may be increased to lower the fluid loadassociated with treatment. Alternatively, delivery vehicles composed ofirritating lipids may be diluted to low concentrations to lesseninflammation at the site of administration. For diagnosis, the amount ofdelivery vehicles administered will depend upon the particular labelused, the disease state being diagnosed and the judgment of theclinician.

Preferably, the pharmaceutical compositions of the present invention areadministered intravenously. Dosage for the delivery vehicle formulationswill depend on the ratio of drug to lipid and the administratingphysician's opinion based on age, weight, and condition of the patient.

In addition to pharmaceutical compositions, suitable formulations forveterinary use may be prepared and administered in a manner suitable tothe subject. Preferred veterinary subjects include mammalian species,for example, non-human primates, dogs, cats, cattle, horses, sheep, anddomesticated fowl. Subjects may also include laboratory animals, forexample, in particular, rats, rabbits, mice, and guinea pigs.

Kits

The therapeutic agents in the invention compositions may be formulatedseparately in individual compositions wherein each therapeutic agent isstably associated with appropriate delivery vehicles. These compositionscan be administered separately to subjects as long as thepharmacokinetics of the delivery vehicles are coordinated so that theratio of therapeutic agents administered is maintained at the target fortreatment. Thus, it is useful to construct kits which include, inseparate containers, a first composition comprising delivery vehiclesstably associated with at least a first therapeutic agent and, in asecond container, a second composition comprising delivery vehiclesstably associated with at least one second therapeutic agent. Thecontainers can then be packaged into the kit.

The kit will also include instructions as to the mode of administrationof the compositions to a subject, at least including a description ofthe ratio of amounts of each composition to be administered.Alternatively, or in addition, the kit is constructed so that theamounts of compositions in each container is pre-measured so that thecontents of one container in combination with the contents of the otherrepresent the correct ratio. Alternatively, or in addition, thecontainers may be marked with a measuring scale permitting dispensationof appropriate amounts according to the scales visible. The containersmay themselves be useable in administration; for example, the kit mightcontain the appropriate amounts of each composition in separatesyringes. Formulations which comprise the pre-formulated correct ratioof therapeutic agents may also be packaged in this way so that theformulation is administered directly from a syringe prepackaged in thekit.

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

EXAMPLES Example 1 Daunorubicin:Cytarabine Synergy In Vitro is DrugRatio Dependent

Many combinations of two or more drugs have the ability to exhibitsynergistic effects. Similarly, combinations of the same two or moredrugs may also show additive or antagonistic interactions. In order toidentify ratios of daunorubicin and cytarabine (also known as, Ara-C)that are synergistic, various combinations of daunorubicin andcytarabine were tested for their cytotoxic effects in vitro. Morespecifically, drug ratios that demonstrate synergy over a broad range ofdrug concentrations were identified.

Measuring additive, synergistic or antagonistic effects was performedusing daunorubicin:cytarabine (DN:Ara-C) at 10:1, 5:1, 1:1, 1:5 and 1:10mole ratios in P388 and L1210 murine lymphocytic leukemia cells. Thestandard tetrazolium-based colorimetric MTT cytotoxicity assay protocol(Mosmann, et al., J. Immunol. Methods (1983) 65(1-2):55-63) was utilizedto determine the readout for the fraction of cells affected. Briefly,viable cells reduce the tetrazolium salt,3-(4,5-diethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to ablue formazan which can be read spectrophotometrically. Cells, such asthe P388 or L1210 murine lymphocytic leukemia cells used here, arepassaged in BDF-1 mice and are removed, as required, and transferredinto 75 cm2 flasks in fresh cell culture medium and added into 96-wellcell culture plates at a concentration of 10,000 or 6,000 P388 or L1210cells per well, respectively, in 100 μL per well. The cells are thenallowed to incubate for 24 hours at 37° C., 5% CO2 and >75% humidity topromote cell adhesion. The following day, serial drug dilutions areprepared in 12-well cell culture plates. The agents, previously preparedin various solutions, are diluted in fresh cell culture media. Agentsare administered to the appropriate wells for single agents (20 μL) andat specific fixed ratio dual agent combinations (increments of 20 μL).The total well volumes are made up to 200 μL with fresh media. The drugexposure is for a duration of 72 hours.

Following drug exposure, MIT reagent (1 mg/mL phosphate buffered saltsolution) is added to each well at a volume of 50 μL per well andincubated for 4 hours. The well contents are then aspirated and 150 μLof dimethylsulfoxide (DMSO) is added to each well to disrupt the cellsand to solubilize the formazan precipitate within the cells. The 96-wellplates are shaken on a plate shaker for a minimum of 2 minutes, and readon a microplate spectrophotometer set at a wavelength of 570 nm. Theoptical density (OD) readings are recorded and the OD values of theblank wells containing media alone are subtracted from all the wellscontaining cells. The cell survival following exposure to agents isbased as a percentage of the control wells cells not exposed to drug.All wells are performed in triplicate and mean values are calculated.

A combination index was then determined for each daunorubicin:cytarabinedose using Calcusyn which is based on Chou and Talalay's theory ofdose-effect analysis, in which a “median-effect equation” has been usedto calculate a number of biochemical equations that are extensively usedin the art. Derivations of this equation have given rise to higher orderequations such as those used to calculate Combination Index (CI). Asmentioned previously, CI can be used to determine if combinations ofmore than one drug and various ratios of each combination areantagonistic (CI>1.1), additive (0.9≦CI≧1.1) or synergistic (CI<0.9). CIplots are typically illustrated with CI representing the y-axis versusthe proportion of cells affected, or fraction affected (fa), on thex-axis. The data in FIGS. 1A and 1B, plotted as CI versus the fractionof P388 or L1210 murine lymphocytic leukemia cells affected (fa),respectively, illustrates that particular combinations of daunorubicinand cytarabine are antagonistic while others are synergistic oradditive. At daunorubicin: cytarabine (DN:Ara-C) ratios of 1:10, 1:5 and1:1, synergy is observed in P388 cells at fa values of 0.75 and above(FIG. 1A). This demonstrates that a 1:1, 1:10 and 1:5 ratio aresynergistic at concentrations sufficient to cause significant tumor cellkill. The 1:5 and 1:10 ratios or daunorubicin:cytarabine are alsonon-antagonistic in L1210 cells over a suitable range of fa values (FIG.1B). In contrast, 5:1 and 10:1 ratios of daunorubicin:cytarabine areantagonistic in P388 and L1210 cells over a broad range of fa values.The dependence of CI on daunorubicin:cytarabine ratio is also presentedin FIGS. 1C and 1D where CI values at drug concentrations sufficient tocause 75% (ED75) and 90% (ED90) tumor cell growth inhibition arecompared at the different daunorubicin:cytarabine molar ratios in P388and L1210 cells. Based on these results, a mole ratio of 1:5daunorubicin:cytarabine was selected for formulating in fixed drug ratioliposome carriers.

Example 2 Daunorubicin and Cytarabine can be Dual-Loaded into Liposomes

Liposomes containing both daunorubicin and cytarabine could be generatedusing DSPC/DSPG/Cholesterol (7:2:1 mole ratio) liposomes containingpassively entrapped cytarabine which were actively loaded withdaunorubicin. Briefly, lipid foams were prepared by dissolving lipids(DSPC:DSPG:CHOL (7:2:1 mol ratio)) mixed at a concentration of 100 mglipid/ml final concentration into a chloroform:methanol: H₂O mixture(95:4:1 vol/vol). The solvent was then removed by vacuum evaporation andthe resulting lipid foams were hydrated with a solution consisting of100 mM Cu(gluconate)₂, 220 mM triethanolamine (TEA), pH 7.4 and 50 mg/mL(203 mM) cytarabine (containing ³H-cytarabine as a tracer) at 70° C. Theresulting MLVs were extruded 10 times at 70° C. to generate largeunilamellar vesicles. The mean diameter of the resulting liposomes wasdetermined by QELS (quasi-elastic light scattering) analysis to beapproximately 100 nm+/−20 nm. Subsequently, the liposomes were bufferexchanged into 300 mM sucrose, 20 mM HEPES, 1 mM EDTA (SHE), pH 7.4,using tangential flow dialysis, thereby removing any unencapsulatedcytarabine and Cu(gluconate)₂/TEA. Cytarabine to lipid molar ratios weredetermined using liquid scintillation counting to determine lipidconcentration (¹⁴C-DPPC) and cytarabine concentration (³H-Cytarabine).

Daunorubicin was added to these liposomes to a final targetdaunorubicin: cytarabine molar ratio of 1:5. Daunorubicin loading intothe liposomes was facilitated by incubating the samples at 50° C. for 30minutes. After loading, the sample was cooled to room temperature. Drugencapsulation efficiency was evaluated after liposome elution through asephadex G-50 spin column.

Daunorubicin loading efficiency was determined using absorbance at 480nm against a standard curve. A drug to lipid ratio at each time pointwas determined using absorbance at 480 nm for daunorubicin measurementand liquid scintillation counting to determine lipid concentrations.Table 1 shows the mean daunorubicin/lipid ratio and cytarabine/lipidratio after drug encapsulation and removal of free drug. It is apparentfrom Table 1 that daunorubicin, added at an initial daunorubicin tolipid ratio of 0.042:1, can be efficiently loaded into DSPC/DSPG/Chol(7:2:1 mole ratio) liposomes, containing passively entrapped cytarabineat a 0.234 drug to lipid ratio.

TABLE 1 Daunorubicin:Cytarabine co-loaded in DSPC:DSPG:CHOL (7:2:1mol:mol) liposomes. Daunorubicin; Cytarabine; Daunorubicin: lipid molarratio lipid molar ratio cytarabine ratio 0.0418 +/− 0.0041 0.234 +/−0.0239 0.178 +/− 0.013 Data represents the mean +/− standard deviation,n = 10.

Example 3 Maintaining Ratios of Drugs In Vivo

To determine if daunorubicin and cytarabine could be maintained at the1:5 drug:drug ratio in the synergistic range in vivo, DSPC/DSPG/Chol(7:2:1 mol:mol), liposomes containing encapsulated daunorubicin andcytarabine were administered intravenously to mice and the plasmadrug/drug ratio was monitored over time.

Briefly, lipid foams were prepared by dissolving lipids mixture(DSPC:DSPG:CHOL (7:2:1 mol ratio)) at a concentration of 100 mg lipid/mlfinal concentration into a chloroform:methanol: H₂O mixture (95:4:1vol/vol). The solvent was then removed by vacuum evaporation and theresulting lipid foams were hydrated with a solution consisting of 100 mMCu(gluconate)₂, 220 mM triethanolamine (TEA), pH 7.4 and 50 mg/mL (203mM) of cytarabine (containing trace amounts of ³H-cytarabine) at 70° C.The resulting MLVs were extruded at 70° C. to generate large unilamellarvesicles. The mean diameter of the resulting liposomes was determined byQELS (quasi-elastic light scattering) analysis to be approximately 100nm+/−20 nm. Subsequently, the liposomes were buffer exchanged into, 300mM sucrose, 20 mM sodium phosphate, 10 mM EDTA, pH 7.4, and then into300 mM sucrose, 20 mM sodium phosphate, pH 7.4, using tangential flowdialysis, thus removing any unencapsulated cytarabine andCu(gluconate)₂/TEA.

Daunorubicin was added to these liposomes such that the daunorubicin tocytarabine final molar ratio would be about 1:5. Daunorubicin loadinginto the liposomes was facilitated by incubating the samples at 50° C.for 30 minutes. After loading, the sample was cooled to roomtemperature. Drug encapsulation efficiency was evaluated after liposomeelution through a Sephadex G-50 spin column. Cytarabine to lipid ratioswere determined using liquid scintillation counting to determine lipidconcentrations (¹⁴C-DPPC) and cytarabine concentrations (³H-cytarabine).Daunorubicin loading efficiency was measured using absorbance at 480 nmagainst a standard curve. The drug to lipid ratio was determined usingabsorbance at 480 nm for daunorubicin measurement and liquidscintillation counting to determine lipid and cytarabine concentrations.

The preparation was then injected intravenously via the tail vein intoBDF-1 mice. Doses of the liposomal formulations were 5 mg/kg ofdaunorubicin and 12.5 mg/kg of cytarabine. At the indicated time pointsafter intravenous administration, blood was collected by cardiacpuncture (3 mice per time point) and placed into EDTA coated microcontainers. The samples were centrifuged to separate plasma, and plasmawas transferred to another tube. Daunorubicin and cytarabine plasmalevels were quantified with High Performance Liquid Chromatography(HPLC).

FIG. 2A shows that plasma elimination curves for daunorubicin andcytarabine at various time points after intravenous administration toBDF-1 mice when they were delivered in the above-described liposomes.One-hour post iv injection the daunorubicin plasma concentration was 167nmol/ml and a concentration of 872 nmol cytarabine/ml plasma wasobserved. At four hours post injection the daunorubicin concentrationwas 143 nmol/ml and the cytarabine concentration was 781 nmol/ml.

FIG. 2B shows that plasma levels of daunorubicin and cytarabine wereeffectively maintained in a synergistic range for extended time afterintravenous administration to BDF-1 mice when the drugs weresimultaneously delivered in the above-described liposomes. Data pointsrepresent the molar ratios of daunorubicin:cytarabine determined inplasma (+/−standard deviation) at the specified time points. Therefore,appropriately designed delivery vehicles such as liposomes can deliverthe desired ratio of daunorubicin and cytarabine in vivo.

Example 4 Daunorubicin and Cytarabine Co-Formulated in Liposomes at aSynergistic Ratio Demonstrates Superior Antitumor Efficacy

To maximize the therapeutic activity of drug combinations and to capturethe synergistic benefits observed in vitro, the drug combination needsto be delivered to the tumors site at the optimal drug to drug ratio. Asingle liposome formulation containing the two drugs at fixed ratiosknown to be synergistic in tissue culture was developed allowingco-ordinate in vivo drug release as illustrated in example 3. Theantitumor activity of this formulation was then evaluated in P388 andL1210 murine lymphocytic leukemia models.

DSPC/DSPG/Chol (7:2:1 mole ratio) liposomes co-encapsulated withdaunorubicin and cytarabine at a synergistic molar ratio ofapproximately 1:5 were prepared as described in Example 3.

In order to perform tumor studies on mice, animals are inoculated ipwith 1×10⁶ P388 or L1210 tumors cells which were then allowed to growfor 24 hr prior to initiation of treatment. Mice were organized intoappropriate treatment groups consisting of control and treatment groupsincluding saline, liposomal Daunorubicin, liposomal Cytarabine, freedrug cocktail and daunorubicin:cytarabine co-loaded in DSPC:DSPG:Chol(7:2:1, mol:mol) liposomes resulting in a final daunorubicin:cytarabinemolar ratio of approximately 1:5. Mice were injected intravenously withthe required volume of sample to administer the prescribed dose to theanimals based on individual mouse weights on days 1, 4 and 7 post tumorcell inoculations. Animals were weighted and monitored for survival andin-life observations are collected at the time of weight measurement.FIG. 3 illustrates the results of these experiments.

As FIG. 3A indicates, a significantly enhanced antitumor activity forthe liposome formulation containing daunorubicin:cytarabine co-loaded atabout a 1:5 molar ratio was observed compared to each single agentformulated individually into liposomes, as well as free drug cocktailadministered at its maximum tolerated dose (MTD). The buffer controlgroup had a median survival time of 8 days. The animals treated withliposomal daunorubicin at a dose of 5 mg/kg exhibited a median survivaltime of 16 days corresponding to an increase in survival time of 100%.The mice treated with liposomal cytarabine at 12.5 mg/kg displayed amedian survival time of 22 days corresponding to an increase in survivaltime of 175% and the mice treated with daunorubicin and cytarabineco-loaded at about a 1:5 molar drug ratio inside DSPC:DSPG:Chol (7:2:1,mol:mol) liposomes exhibited a median survival time of >60 days andincrease in life span of >650% with 9/10 long term survivors. Incomparison, the mice treated with the daunorubicin:cytarabine as a freedrug cocktail at its MTD (based on maximizing dose of both free drugs)did not respond as well to the antitumor therapy as reflected by themedian survival time of 27 days and the corresponding increase in lifespan of 237%.

Similarly, as seen in FIG. 3B, superior antitumor activity was achievedfor the liposome formulation with daunorubicin and cytarabine co-loadedat about a 1:5 molar ratio as compared to either the corresponding freedrug cocktail or each drug loaded in a liposome individually. The buffercontrol group had a median survival time of 7 days. Mice treated witheither liposome-encapsulated daunorubicin or liposome-encapsulatedcytarabine had a median survival time of 20 and 43.5 days, respectively.In comparison, the animals treated with daunorubicin and cytarabineco-loaded at a molar ratio of about 1:5 into DSPC/DSPG/Chol (7:2:1 molratio) liposomes exhibited a median survival time of greater than 60days.

These results demonstrate that fixing synergisticdaunorubicin:cytarabine ratios by encapsulating them insideappropriately designed liposomes can dramatically improve antitumoractivity.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents.

The invention claimed is:
 1. A composition for parental administrationto a subject which composition comprises liposomes having associatedtherewith daunorubicin and cytarabine, and wherein (a) the daunorubicinand cytarabine are co-encapsulated; (b) the daunorubicin:cytarabine moleratio is about 1:5; (c) the liposomes have a mean diameter of less than250 nm; and (d) wherein the liposomes comprise DSPC, DSPG andcholesterol in a mole ratio of about 7:2:1.
 2. A method to treat aleukemia in a subject which comprises administering to said subject aneffective amount of the composition of claim
 1. 3. The method of claim2, wherein the subject is a human.
 4. The method of claim 2, wherein thesubject is a non-human mammal or avian.
 5. The method of claim 3,wherein the leukemia is ALL or AML.
 6. A composition for parentaladministration to a subject which composition comprises liposomes havingassociated therewith daunorubicin and cytarabine, and wherein (a) thedaunorubicin and cytarabine are co-encapsulated; (b) thedaunorubicin:cytarabine mole ratio is about 1:5; (c) the compositioncomprises no therapeutic agents in addition to the daunorubicin andcytarabine; and (d) wherein the liposomes comprise DSPC, DSPG andcholesterol in a mole ratio of about 7:2:1.
 7. A method to treat aleukemia in a subject which comprises administering to said subject aneffective amount of the composition of claim
 6. 8. The method of claim7, wherein the subject is a human.
 9. The method of claim 7, wherein thesubject is a non-human mammal or avian.
 10. The method of claim 8,wherein the leukemia is ALL or AML.